The nuclear transplantation involves the transplantation of donor cells or cell nuclei into enucleated oocytes. The resultant nt-units can develop to various pre-implantation stages, or further into life-borne animals. This method was shown to be successful when applied to amphibians at the end of the 1950s. Briggs and King obtained nuclear transferred frogs by transferring nuclei of the enteric epithelium of the rananigromaculata into oocytes. Nuclear transfer had not been applied to mammals until the end of 1980s. Many types of somatic cells were used in the nuclear transfer experiments as the nuclear donor cells, including embryo blastomeres, inner cell mass, and terminal embryo cells in nuclear transfer (Collas et al., Mol. Reprod. Dev., 38:264-267, 1994; Keefer et al., Biology of Reproduction, 50:935-939, 1994; Sims et al., PNAS, 90:6155-6159, 1993).
Using adult sheep mammary gland as donor cell, Wilmut et al. in Britain (Nature 1997, 385, 810-813) produced the first living lamb from somatic cell nuclear transfer. In 1998, sequential mice somatic cells nuclear transplantation into was successfully completed in US (Wakayama, et al. Nature 394: 369-374, 1998). In 1999, nuclear transplantation of mice embryonic stem cell (ES) was completed (Teruhiko et al., PNAS 96:14984-14989, 1999). The success of nuclear transplantation using adult somatic cells is not only a progress in technique but also a progress in concept, showing the possibility that highly differentiated adult somatic cell nuclei can form new individuals once being reprogrammed to reenter development
In 1999, Dominko et al. injected somatic cell nuclei from various animals (e.g. cows, sheep, pigs, monkeys, and rats) into bovine oocytes to develop nt-units, each developing to some extent (Biology of Reproduction. 60 (6): 1496-1502, 1999). These experiments show that mammalian somatic nuclei can be activated by oocytes of a species different from the nuclear donor to form nt-units. Such nt-units can develop to all the pre-implantation stages. The finding, that oocytes of one species can reprogram somatic nuclei of another species, shows that mechanisms controlling reprogramming are highly conserved in different mammalian species.
The advancement in ES cell cultivation has been also highlighted all over the world these years while the development of the nuclear transfer technology is flourishing. The basic manipulation involved in the establishment of ES cell and the basic characters and the application thereof have been well known in the art since the establishment of the mice ES cell line in 1981 (See Evans, et al. Nature, 29: 154-156, 1981; Martin, et al. PNAS, 78: 7634-7638, 1981). The ES cell can be kept in an undifferentiated, infinitely proliferating state. providing that the cultivation thereof is effected in a feeder layer of fibroblast cells (Evans, et al.) or under differentiation inhibiting conditions (Smith, et al. Development Biology, 121:1-9, 1987).
ES cells have the potential of development into all cell types of a body, including germ cell. ES cells can be differentiated to various specific cell types under appropriate induction conditions. Embryonic stem cells have been successfully directed to differentiate in vitro into various types of cells, e.g. the hematopoletic stem cells (Ronald, et al. PNAS, 92: 7530-7534, 1995), neural cells (Dinsmore, et al. Theriogenology, 49: 145-151, 1998), muscle cells (Reubinoff, et al. Nature Biotechnology, 18 (4): 399-404, 2000), adipocytes (Dani C Smith, et al. J Cell Sci, 110: 1279-1285, 1997), endothelial cells (Vittet, et al. Blood, 88 (9): 3424-3431, 1996) and so on. A specific cell type, e.g. a muscle-like cell, differentiated from ES cells display properties similar to that of its natural equivalent cell types, e.g. a muscle cell, therefore, cells differentiated from ES cell can be use in treatment of diseases (cell, tissue, or organ transplantation).
In view of the potentiality of the mouse ES cells, it has been tried to culture the ES cells of large mammals because the establishment thereof not only has the significance in scientific research but also can be applied to medicine. For example, the human ES cells can be directed to all kinds of specialized cells for the treatment of diseases. Because of their proliferation and differentiation potential, ES cells provided a platform for genetic modification. ES cells of large animals can be genetically modified to produce various biological products.
Isolation of ES cells or embryonic stem-like cells from large mammals have been reported. For example, Notarianni, et al. (J. Reprod. Fert., Suppl. 43: 255-260, 1991) reported that the cells in primary cultures of inner cell masses from pig and sheep blastocysts exhibit some morphological and growth characteristics similar to ES cells. Chen R L, et al. (Biology of Reproduction, 57 (4): 756-764, 1997) and Wianny, et al. (Theriogenology, 52 (2): 195-212, 1999) reported the isolation of pig ES cells from porcine blastocysts, respectively,
Stekelenburg-Hamers, et al. reported the isolation and the characterization of embryonic stem-like cells from inner cell mass of bovine blastocysts (Mol. Reprod. 40: 444-454, 1995).
Thomson, et al. reported the successful isolation of ES cells from primate macaque (PNAS, 92 (17): 7844-8, 1995).
Thomson, et al. successfully established human ES cells lines (Science, 282 (6): 1145-1147, 1998), which is an important breakthrough in the stem cells research. These call lines not only can be used as important tools in the research of human development, but also has the broad application prospect in medical fields. For example, (1) human ES cells lines can be expanded and differentiated into specific cell types to meet the needs of the patients. They will become the cell source for cell or organ transplantation therapies. It is possible that many human diseases can be treated through cell transplantation. Besides its medical applications, (2) human ES cell lines may also facilitate the screen for new drugs and the safety evaluation of drugs.
However, the cells differentiated from the human ES cells may cause immune rejection while being used in the transplantation between the individuals of different MHC types, thus the patient would have to take immune inhibitor, which is toxic. At present, there is no method in obtaining ES cells that are compatible with the patient's immune systems by using his somatic cells.
Munsie, at al. reported the isolation of mice ES cells from blastocysts derived by somatic cell nuclear transfer (Current Biology 10: 989-992, 2000). Wakayama, et al. obtained the mice ES cells, which can be induced to various types of specific cells in vitro, from the cultures of blastocysts derived by somatic cell nuclear transfer (Science, 292 (5517); 740-743. 2001). The result of the research done by Wakayama, et al. demonstrates that ES cells can be isolated from nuclear transfer embryos by somatic cell nuclear transfer. The ntES cells of somatic cell origin are pluripotent and can differentiate into any specific cell types as ES cells derived from the normal zygote.
The successes achieved by all of these scientists mentioned above established a new route for treatment of the diseases, i.e., therapeutic cloning. It is suggested that somatic cells of the patient can be reprogrammed through nuclear transfer to produce ntES cells. The ntES cells obtained are further differentiated into the specific cell type needed by the patient. ntES cells and their differentiated progenies have the same genotype as the patient, and therefore would not be rejected by the patient's immune system when transplanted to the patient. Therapeutic cloning provides an approach to solve the problem of immune rejection commonly observed in transplantation medicine.
WO 98/07841 (Robe, et al., Massachusetts, U.S.A., filed in 1998) disclosed the isolation of the thirty 2-cell-stage embryos and six 4- to 16-cell-stage embryos and one 16- to 400-cell-stage embryo from the allogeneic nuclear transplantation from lymphocyte and mouth epithelium of human to the bovine oocytes. However, this study failed to provide any proof demonstrating that the embryos and cell colonies derived from the embryos were encoded by human genomic DNA rather than bovine genomic DNA. Blastocysts can be easily created through parthenogenesis of bovine oocytes. Furthermore, the patent application provided no proof demonstrating that the colonies were encoded by human genomic DNA and displayed any characteristics of human or primate stem cells,
Up to the submission of the present application, there has been no report that the ntES cells can be obtained by human somatic cell nuclei transfer, neither the report that human specific cell types can be differentiated therefrom.